This application relates to bacteriocin compositions for use as enhanced broad range bactericides and methods of preventing and treating microbial infection.
Bacteriocins such as lysostaphin and nisin are proteins produced by bacteria that inhibit the growth of and sometimes kill bacteria closely related to the species of their origin. Lysostaphin is a bacteriocin that lyses and kills practically all known species of Staphylococcus, but is inactive against bacteria of other genera. Lysostaphin, isolated from culture filtrates of Staphylococcus simulans (NRRL B-2628) grown according to published references, is an endopeptidase which cleaves the polyglycine cross-links of the peptidoglycan found in the cell walls of Staphylococcus. Cultures of S. simulans grown under conditions which induce the production of lysostaphin are immune to the bacteriocin while the same cultures grown under conditions whereby lysostaphin is not produced are sensitive to the bacteriocin.
Lysostaphin is a naturally occurring bacteriocin secreted by a single known strain of S. simulans originally isolated and named Staphylococcus staphylolyticus by Schindler and Schuhardt. The production of lysostaphin by S. staphylolyticus has been described previously in U.S. Pat. No. 3,278,378 issued Oct. 11, 1966 and in Proceedings of the National Academy of Sciences, 51:414-421 (1964). The single organism S. staphylolyticus (NRRL B-2628) which produced lysostaphin was recently identified as a biovar of S. simulans by Sloan et al., Int. J. System. Bacteriol., 32:170-174 (1982). Since the name S. staphylolyticus is not on the Approved List of Bacterial Names, the organism producing lysostaphin has been redesignated as S. simulans.
Previously it was shown that the action of lysostaphin can be potentiated by penicillin and other antibiotics. See copending U.S. application No. 188,183 to Blackburn et al. filed Apr. 28, 1988.
Nisan, although sometimes referred to as a peptide antibiotic is more properly referred to as a bacteriocin. Nisin is produced in nature by various strains of the bacterium Streptococcus lactis. It is a food preservative used to inhibit the outgrowth of spores of certain species of Gram positive bacilli, including those arising from strains of Clostridium known to be responsible for Botulism food poisoning. A summary of nisin's properties appears in Hurst, Advances in Applied Microbiology, 27:85-123 (1981). The publication describes what is generally known about nisin. Nisin, produced by Streptococcus lactis, is commercially available as an impure preparation, Nisaplin.TM., (Aplin & Barret Ltd., Dorset, England)
Nisin belongs to the class of peptides containing lanthionine. Also included in that class are subtilin, epidermin, cinnamycin, duramycin, ancovenin, and Pep 5. These bacteriocin peptides are each produced by different microorganisms. However, subtilin obtained from certain cultures of B. subtilis, and epidermin obtained from certain cultures of Staphylococcus epidermidis, have molecular structures very similar to that of nisin, Hurst, pp. 85-86; and Schnell et al. Nature 333:276-278. Structurally similar, lanthionine containing peptide bacteriocins are believed to be effective in place of nisin in the present invention.
Nisin has been applied effectively as a preservative in processed cheese, and dairy products. The use of nisin in processed cheese products has been the subject of recent patents. See U.S. Pat. Nos. 4,584,199 and 4,597,972. The use of nisin to inhibit the outgrowth of certain Gram positive bacterial spores has been well documented. See Taylor, U.S. Pat. No. 5,584,199, and Taylor, U.S. Pat. No. 4,597,972, Tsai and Sandin, "Conjugal Transfer of Nisin Plasmid Genes from Streptococcus lactis 7962 to Leuconostoc dextranicum 181", Applied and Environmental Microbiology, p. 352 (1987); "A Natural Preservative", Food Engineering International, pp. 37-38 (1987); "Focus on Nisin", Food Manufacture, p. 63 (1987). Nisin is sometimes found naturally-occurring in low concentration in milk and cheese, and is believed to be completely non-toxic and non-allergenic to humans. Nisin has recently been recognized as safe by the FDA as a direct food ingredient in pasteurized cheese spread, pasteurized processed cheese spread and pasteurized or pasteurized processed cheese spread with fruits, vegetables, or meats. As nisin is proteinaceous, any residues in ingested foods are quickly degraded by digestive enzymes.
The general acceptance of nisin as a food preservative has been limited by the teaching that, as a bacteriocin, the activity of nisin was restricted to include only those Gram positive bacteria closely related to the bacterial species of its origin. Furthermore, nisin has not previously been shown to have bactericidal activity towards Gram negative bacteria. Since food contamination and spoilage result from a diversity of Gram positive and Gram negative bacteria, it is not surprising, therefore, that nisin has received only limited acceptance as a food preservative. Moreover, because of the heretofore restricted activity of nisin as a bacteriocin, its uses as such outside of the food area have not been indicated.
It has recently been demonstrated that a composition comprising nisin and non-bactericidal agents such as chelating agents and surfactants has bactericidal activity towards a wide range of Gram negative bacterial species and enhanced activity towards a broad range of Gram positive bacterial species. For instance Gram negative bacteria shown to be sensitive to the enhanced bactericide are Salmonella typhimirium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacterioides gingivalis and Actinobacillus actinomycetescomitans. Gram positive bacteria shown to be sensitive to the enhanced bactericides are Staphylococcus aureus, Streptococcus mutans, Listeria monocytogenes, Streptococcus agalactiae and coryneform bacteria. See copending Blackburn et al., U.S. patent application entitled Nisin Compositions For Use as Enhanced, Broad Range Bactericides which is a continuation-in-part of U.S. patent application Ser. No. 209,861 filed June 22, 1988 which is hereby incorporated herein by reference.